Human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) are the etiologic agents of acquired immunodeficiency syndrome (AIDS), which results from the profound depletion of CD4-positive lymphocytes in infected individuals (Barre-Sinoussi, F., Science 1983; Gallo, R. C., et al., Science 1984; Fauci, A. S., et al., Ann Intern Med 1984).
The entry of HIV-1 into target cells is mediated by the viral envelope glycoproteins. The exterior envelope glycoprotein, gp120, and the transmembrane envelope glycoprotein, gp41, are derived from a gp160 precursor (Earl, P. L., et al., J Virol 1984). The gp160 glycoprotein results from the addition of N-linked, high mannose sugar chains to the approximately 845-870 amino acid primary translation product of the env gene in the rough endoplasmic reticulum (ER) [Ibid.]. Oligomers of gp160 form in the endoplasmic reticulum, but the current data do not unambiguously distinguish whether trimers or tetramers constitute this higher-order complex (Earl, P. L., Proc Natl Acad Sci 1987; Pinter, A., et al., J Virol 1989; Schawaller, M., et al., Virology 1989; Lu, M., et al., Nat Struct Biol 1995). Early results studying cell- or virion-associated HIV-1 envelope glycoproteins suggested the formation of dimers, followed by the assembly of dimers into unstable tetramers (Earl, P. L., Proc Natl Acad Sci 1987; Pinter, A., et al., J Virol 1989). This interpretation was supported by the analysis of soluble forms of gp160 lacking a membrane-spanning region (Schawaller, M., et al., Virology 1989). By contrast, studies of peptide fragments of the gp41 ectodomain, which was shown to be necessary of the oligomerization of soluble forms of gp160, revealed a strong tendency for trimer formation (Lu, M., et al., Nat Struct Biol 1995). More recent structural studies of these gp41 peptides have revealed a trimeric coiled coil (Chan, et al. Cell 899: 263-273 (1997); Weissenhorn et al. Nature 384:184-187 (1997)).
HIV-1 infects T lymphocytes, monocytes/macrophage, dendritic cells and, in the central nervous system, microglia (Gartner et al., 1986; Koenig et al., 1986; Pope et al., 1994; Weissman et al., 1995). All of these cells express the CD4 glycoprotein, which serves as the receptor for HIV-1 and HIV-2 (Dalgleish et al., 1984; Klatzman et al., 1984; Maddon et al., 1986). Efficient entry of HIV-1 into target cells is dependent upon binding of the viral exterior envelope glycoprotein, gp120, to the CD4-amino-terminal domain (McDougal et al., 1986; Helseth et al., 1990). After virus binding, the HIV-1 envelope glycoproteins mediate the fusion of viral and host cell membranes to complete the entry process (Kowalski et al., 1987; Stein et al., 1987; Helseth et al., 1990). Membrane fusion directed by HIV-1 envelope glycoproteins expressed on the infected cell surface leads to fusion with uninfected CD4-positive cells, resulting in syncytia (Lifson et al., 1986; Sodroski et al., 1986).
Host cell factors in addition to CD4 are necessary for effective HIV-1 envelope glycoprotein-mediated membrane fusion. Some human and animal cells have been shown to be resistant to HIV-1 infection and syncytium formation even when human CD4 was expressed on the cell surface (Maddon et al., 1986; Ashorn et al., 1990; Chesebro et al., 1990; McKnight et al., 1994). Experiments with somatic cell hybrids suggested the possibility that a positive factor expressed in cells susceptible to syncytium formation could complement he block to fusion in resistant cell types (Clapham et al., 1991; Dragic et al., 1992; Broder et al., 1993). HIV-1 variants exhibiting distinct differences in the ability to fuse with and to enter particular subsets of CD4-positive cells have been identified (Broder and Berger, 1995).
All primary clinical HIV-1 isolates, defined as viruses that have not been passaged on immortalized cell lines, replicate in primary monocytes/macrophages and in primary T lymphocytes. Two groups of primary HIV-1 isolates have been defined, based on replication rate in peripheral blood mononuclear cells (PBMC) and the ability to infect and induce the formation of syncytia in immortalized CD4-positive cell lines (Asjo et al., 1986; Cheng-Mayer et al., 1988; Fenyo et al., 1988; Tersmette et al., 1988).
Most primary HIV-1 viruses that initiate human infection and that persist throughout the course of infection replicate to low levels in PBMC and do not replicate in immortalized T cell lines (Asjo et al., 1986; Schuitemaker et al., 1991; Schuitemaker et al., 1992; Connor et al., 1993, 1994a,b). These viruses are referred to herein as macrophage-tropic primary isolates (sometimes referred to as xe2x80x9cMxe2x80x9d). In some HIV-1-infected individuals, viruses that replicate to higher levels in PBMC and that can infect and induce the formation of syncytia in immortalized CD4-positive cell lines emerge late in the course of infection (Asjo et al., 1986; Schuitemaker et al., 1992; Connor et al., 1993, 1994a,b). These viruses will be referred to herein as T cell line-tropic primary viruses (sometimes referred to as xe2x80x9cTxe2x80x9d) The T cell line-tropic primary viruses, by virtue of their ability to replicate on some immortalized cell lines, serve as precursors to the laboratory-adapted isolates, which have been extensively passaged on such cell lines. Laboratory adaptation, however, results in a loss of the ability of HIV-1 to replicate in primary monocyte/macrophage cultures (Schuitemaker et al., 1991; Chesebro et al., 1991; Westervelt et al., 1992; Valentin et al., 1994). Thus, while all HIV-1 isolates replicate on primary T lymphocytes, three groups of virus variants can be defined based on the ability to replicate in primary monocyte/macrophages or in immortalized T cell lines: (1) macrophage-tropic primary viruses that cannot infect T cell lines; (2) laboratory-adapted viruses that cannot infect primary monocytes/macrophages; and (3) T cell line-tropic primary viruses that exhibit dual-tropism for these cell types.
Changes in the viral envelope glycoproteins, in particular in the third variable (V3) region of the gp120 exterior envelope glycoprotein, determine tropism-related phenotypes (Cheng-Mayer et al., 1990; O""Brien et al., 1990; Hwang et al., Westervelt et al., 1992; Chesebro et al., 1992; Willey et al., 1994). Amino acid changes in the V3 region (Helseth et al., 1990; Freed et al., 1991; Ivanoff et al., 1991; Bergeron et al., 1992; Grimaila et al., 1992; Page et al., 1992; Travis et al., 1992) and the binding of antibodies to this domain (Putney et al., 1986; Goudsmit et al., 1988; Linsley et al., 1988; Rusche et al., 1988; Skinner et al., Javeherian et al., 1989) have been shown to disrupt a virus entry process other than CD4 binding. The dependence of the phenotype resulting from V3 structural variation on the particular target cell suggested that the V3 region, which contains a surface-exposed, disulfide-linked loop (Leonard et al., 1990; Moore et al., 1994), might act in conjunction with target cell moieties to determine the efficiency of membrane fusion events.
A G protein-coupled seven transmembrane segment receptor, variously called HUMSTR, LCR-1 or LESTR now referred to as CXCR4 (Federsppiel et al., 1993; Jazin et al., 1993; Loetscher et al., 1994) has been shown to allow a range of non-human, CD4-expressing cells to support infection and cell fusion mediated by laboratory-adapted HIV-1 envelope glycoproteins (Feng et al., 1996). Antibodies to HUMSTR blocked cell fusion and infection by laboratory-adapted HIV-1 isolates but not by macrophage-tropic primary viruses (Feng et al., 1996). While its natural ligand is currently unknown, HUMSTSR exhibits sequence similarity to the receptor for interleukin-8, an alpha (CXC) chemokine) (Probst et al., 1992). Other G-protein-coupled seven transmembrane segment receptors such as CCR5, CCR3 and CCR2 have been shown to assist cellular entry of other HIV-1 isolates. It is believed that the cellular entry occurs as a result of the interaction of gp120, CD4 and the chemokine receptor.
These discoveries emphasize the significant role env plays in viral entry. And they further illustrate the importance of env as a target in inhibiting the spread of infection. However, attempts at targeting env have not been as successful as hoped. For example, early attempts were made to develop vaccines based upon using a subunit approach, which focuses on using less antigens then present in the entire virus, because of the significant health concerns raised in using attenuated or inactivated whole HIV because of the severity of HIV infection. A key subunit vaccine target was the envelope glycoprotein. However, these attempts at developing a subunit vaccine using the env were not successful. Even generating antibodies to env that can neutralize a wide range of HIV strains initially presented many difficulties. While considerable improvement has occurred in understanding how to generate antibodies to env, e.g. gp120 antibodies; such as by using gp120 conformational polypeptides where portions of the variable regions have been eleted, further improvements would be useful.
We have discovered DNA sequences encoding env, where we can introduce sequences encoding cysteine residues in a portion encoding the gp 41 transmembrane envelope glycoprotein. These sequences will express proteins that can stably oligomerize in a conformation approaching the native virus. The introduction of these residues creates the molecular contacts between alpha helices that stabilize the trimeric coiled coil, which is responsible for the oligomerization of the HIV-1 envelope glycoprotein. These cysteine residues are introduced in specific locations along these alpha helices. One preferred location is at the residues adjacent to the d and e positions of the coiled coil helix such as positions 576 and 577 of HIV-1. It is also preferred that an adjoining amino acid residue be substituted to provide greater flexibility in the protein backbone; one example is the substitution of a gly at the f position such as 578 of HIV-1. As a result of these changes, the normally labile HIV-1 gp160 envelope glycoprotein was converted into a stable disulfide-linked oligomer that was expressed on the cell surface and had a conformation approaching that of the native glycoprotein as demonstrated by its ability to be recognized by a series of conformationally dependent antibodies. The pattern of hetero-oligomer formation between this construct and an analogous construct lacking portions of the gp120 variable loops and of the gp41 cytoplasmic tail demonstrates that these oligomers are trimers. The stabilized oligomer can be used to generate a range of antibodies that recognize and interact with a diverse range of HIV strains. The DNA sequence can also be used as a subunit vaccine.